Human and animal cells are exposed to a variety of causes of DNA damage such as reactive oxygen species, UV light, x-rays and endogenous or exogenous cytostatic agents.
Cytostatic agents are agents which inhibit or suppress cellular growth and/or multiplication (proliferation/replication), for example by damaging DNA or by interfering with the cellular replication machinery. Alkylating agents are a class of cytostatic agents, some of which are used clinically or for research purposes.
Alkylating agents cause DNA damage by modifying bases at N or O atoms. The type of damage depends on the type of agent, with most agents causing a specific DNA modification. DNA damage includes alkylation adducts and inter strand cross-links which may lead to miscoding during replication and/or replication blocks followed by double strand breaks or translesion synthesis.
Human and animal cells possess various DNA repair systems including base excision repair, nucleotide excision repair and mismatch repair. An example is the human DNA oxidative demethylase, hABH2, which reconverts 3-methylcytosine (3meC) into cytosine and 1-methyladenine (1meA) into adenine by oxidative demethylation.
Close co-ordination between DNA repair and cell-cycle regulated DNA replication is essential for genome integrity. It is important that in the presence of damage, DNA replication is halted until the damage has been repaired, otherwise mutations arise and are propagated. One protein known to be involved in both DNA replication and DNA repair is proliferating cell nuclear antigen (PCNA).
PCNA is member of the sliding clamp family of proteins which are functionally conserved from bacteria to higher eukaryotes, and whose main function is to provide replicative polymerases with the high processivity needed for duplication of the genome. In live S-phase cells, PCNA tagged with green fluorescent protein (GRP) forms distinct foci representing sites of replication. It can therefore be used as an S-phase marker.
Numerous proteins involved in cellular processes such as DNA repair, chromatin assembly, epigenetic and chromatin remodelling, sister-chromatid cohesion, cell cycle control and survival are localised in so-called replication factories which contain more than a dozen replication forks. Many of these proteins interact with PCNA through the conserved PCNA interacting peptide sequence called the PIP-box (QxxL/I/MxxF/DF/Y), wherein x can be any amino acid. An alternative PCNA binding motif called the KAx box was identified using a peptide display library, but this motif has not been verified to be important for PCNA interactions in vivo.
Various proteins interact with PCNA and some of these proteins, including hABH2, have been shown to co-localise with PCNA in replication foci. However, co-localisation in itself does not imply that there is any direct or indirect interaction between co-localising proteins. Indeed, the absence in hABH2 of PCNA-binding motifs such as the PIP-box or the KAx box would suggest that hABH2 does not interact with PCNA.